The objective of this project is to study in depth the structure and function of two Fe-S proteins: aconitase and a 7Fe ferredoxin. Aconitase is a large enzyme which employs a [4Fe-4S] cluster to catalyse stereospecific dehydration/rehydration of citrate in the second and third steps of the Krebs cycle. Aconitase is the best studied example of a Fe-S enzyme catalysing a non-redox reaction. The goal is to deduce specific steps in the mechanism and the role of the protein and cluster in each. The 7Fe ferredoxin from Azotobacter vinelandii functions in electron transfer reactions and contains [3Fe-4S] and [4Fe-4S] clusters in 106 amino-acids. This protein provides an ideal system for understanding the inter-relationship of polypeptide and Fe-S cluster. Both aconitase and ferredoxin will be studied using x-ray crystallography coupled with -molecular biology, biochemistry and spectroscopy. Each part of the project involves a close collaboration. The P.I. (Stout) will solve, refine and analyze crystal structures of aconitase complexed with substrates,inhibitors and reaction intermediate analogs, and structures of aconitase mutants prepared by the Co-P.I. (Zalkin). Zalkin will carry out mutational analysis of porcine aconitase directed toward active site residues, Fe-S cluster ligands,the hinge/linker region and the leader sequence. Aconitase mutants will be expressed in yeast to study in vivo the effect of mutations, and the mechanisms of mitochondrial import and Fe-S cluster assembly. Samples of bovine aconitase and its substrates will be provided by, and mutant enzyme activity and spectroscopic analyses will be done by collaborators H. Beinert and M. C. Kennedy as part of their continuing study of the enzyme. The P.I. will solve, refine and analyze crystal structures of site-directed mutants of the 7Fe ferredoxin. These proteins will be prepared by B. K. Burgess as part of her on-going mutational analysis of structure and function of this ferredoxin.